Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

Stresser, David M.; Turner, Stephanie D.; Blanchard, Andrew P.; Miller, Vaughn P.; Crespi, Charles L.

doi:10.1124/dmd.30.7.845

Cited by 151 publications

(111 citation statements)

References 21 publications

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“…Inhibition of P450 3A4 activity was monitored using a standard fluorescence-based assay (30), and depletion of NADPH was followed by monitoring the absorbance at 340 nm. NADPH was not rate limiting in incubations with the inhibitors, as determined by quantifying NADPH depletion as a function of incubation time.…”

Section: Methodsmentioning

confidence: 99%

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

Bateman

Baker

Wilke

et al. 2004

Chem. Res. Toxicol.

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Protein covalent labeling can be an undesirable property of compounds being studied in drug discovery programs. Identifying such compounds relies on the use of radiolabeled material, which requires an investment in time and resources not typically expended until later in the discovery process. We describe the detection of covalent adducts to cytochrome P450 3A4, the most abundant and important P450 from a human and drug discovery viewpoint, using liquid chromatography mass spectrometry. The technique is illustrated using L-754,394 and 6′,7′-dihydroxybergamottin, two known inhibitors of P450 3A4. Mass spectrometry of the intact apoprotein as well as the adducted protein is demonstrated. Such methodology may provide the means for screening compounds for covalent protein binding without the use of a radiolabel. It also provides direct information about mechanism-based inhibitors in terms of extent, stoichiometry, and nature of the adduct(s) (mass shift). This information may provide a means for understanding the mechanism of covalent labeling earlier in a drug discovery environment.

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Section: Methodsmentioning

confidence: 99%

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

Bateman

Baker

Wilke

et al. 2004

Chem. Res. Toxicol.

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“…Different CYP3A substrates exist for the incubation step, but all of them are substrates for human CYP3A. In the present study, we decided to work with the human CYP3A substrate BFC, which is catalyzed by CYP3A and small contribution of CYP1A2 [30,31], that is converted into a fluorescent product by CYP3A. We first wanted to know whether this human CYP3A substrate is also metabolized by CYP3A enzymes of different fish.…”

Section: Discussionmentioning

confidence: 99%

“…We first wanted to know whether this human CYP3A substrate is also metabolized by CYP3A enzymes of different fish. For this reason, we adapted the protocol from Mensah-Osman et al [29] to evaluate whether the human-specific CYP3A substrate BFC [30][31][32] can also be catalyzed through fish CYP3A enzymes into the fluorescent product, as in carp [24]. As shown in Figure 1, fish CYP3A enzymes indeed can catalyze BFC.…”

Section: Discussionmentioning

confidence: 99%

A microtiter‐plate‐based cytochrome P450 3A activity assay in fish cell lines

Christen

Oggier

Fent

2009

Enviro Toxic and Chemistry

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Abstract-Enzymes belonging to the cytochrome P450 3A (CYP3A) subfamily play an important role in the metabolism of endogenous substances and xenobiotics, including pharmaceuticals. Xenobiotics can alter CYP3A expression and activity, and therefore, changes in CYP3A activity may serve as a biomarker of xenobiotic exposure. To determine changes in CYP3A enzyme activity for environmental risk assessment of xenobiotics including pharmaceuticals, high-throughput assays are needed, but these are missing for fish cells to date. Here, we report on the development of a fluorescent-based CYP3A high-throughput assay for four fish cell lines cultivated in 96-well plates based on 7-benzyloxy-4-trifluoromethylcoumarin as a CYP3A substrate. We show that human CYP3A substrate BFC is catalyzed by fish CYP3A enzymes to a fluorescent product. Its formation is dependent on cell numbers and incubation time. Furthermore, we demonstrate that with this new CYP3A assay induction and inhibition of enzyme activity by pharmaceuticals can be determined. This new cell-based assay is suitable for detection of alteration in CYP3A enzyme activity in largescale experiments for screening of pharmaceuticals occurring in the environment.

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“…This probe has been shown to be highly selective for rat CYP2D2, as well as the human CYP2D6 isoform. 48,56 Rat hepatic microsomes were used in place of the purified human CYP2D6 utilized in the in vitro assay. Assays were performed as described above.…”

Section: Inhibition Studies Purified Cyp2d6mentioning

confidence: 99%

Interactions between Over-the-Counter and Illicit Drugs Utilizing Cytochrome P450 Metabolism: Potential for Exacerbation of Pharmacological Response

Wallace¹,

Crosswy²

2017

Toxicol Forensic Med Open J

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Citation ResearchAbSTRACT Aim: To determine the interaction of over-the-counter (OTC) and illicit psychostimulants at the cytochrome P450 enzyme, CYP2D6. CYP2D6 is responsible for 20% of hepatic Phase I metabolism and is a site of drug-drug interactions, leading to increased drug toxicity. Materials and Methods: We examined the effects the OTC drugs; 1) the prototype H 2 -antagonist cimetidine (CMT) and 2) the opioid agonist cough suppressant dextromethorphan (DEX); as well as two scheduled drugs, methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) for their ability to interfere with CYP2D6 activity. Assays with human CYP2D6 determined the inhibitory potential (IC 50 ) of each drug. Kinetic analysis (V max and K m ) was accomplished using rodent hepatic microsomes. Results: Maximum inhibition of CYP2D6 activity following exposure to CMT+MDMA was significantly reduced 75-85% compared to quinidine (control) values. These data showed inhibitory effects in CYP2D6 activity in each compound tested. Alterations in CYP2D6 activity may result in complex drug-drug interactions leading to elevated plasma levels of drugs and increased risk for toxicity. Assays using rat CYP2D2 demonstrated V max elevations in the CMT group (493%) compared to control (naïve, no treatment) values (19.9±5.1 pmol/mg protein/ min). The K m was increased 218% in CMT compared to controls (3.1±0.5 μM). Collectively, all MA challenged groups exhibited increases in total enzyme [V max ; 280-490%] and affinity [K m ; 165-220%] values compared to the control group. The increase in both V max and K m suggests that the low affinity/high capacity CYP2D2 isoform is upregulated. Conclusion: Our findings suggest that in vivo, MA acts as a CYP2D2-inducer, which will lead to altered secondary drug metabolism, increasing the risk of drug-related toxicity. Coupled with the ability of CMT and DEX to interfere with MA metabolism, a complex drug-drug interaction is possible, leading to increased toxicity. Our findings substantiate the hypothesis that the combination of illicit and OTC drugs could result in complex drug-drug interactions increasing the risk for severe drug-related toxicity.

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Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

Cited by 151 publications

References 21 publications

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

A microtiter‐plate‐based cytochrome P450 3A activity assay in fish cell lines

Interactions between Over-the-Counter and Illicit Drugs Utilizing Cytochrome P450 Metabolism: Potential for Exacerbation of Pharmacological Response

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